Proteases are enzymes that hydrolyze peptide bonds which covalently link amino acids together to form proteins. The presence of proteases in biological samples often causes serious problems in protein purification and in long-term storage of protein samples. These proteases hydrolyze the peptide bonds of the proteins, creating new impurities during the protein purification procedure. Protease contamination in biological samples preserved for long-term storage often causes decomposition of the sample.
Many proteases are classified as "serine" proteases. This means that an unusually reactive serine is part of the active site in the protease enzyme. It is known that the unusually reactive serine in the active site of the protease can be specifically labelled with an organic fluorophosphate such as diisopropylphosphofluoridate (DIPF). Another name for DIPF is diisopropylfluorophosphate. The DIPF reacts with the serine in the active site to form an inactive diisopropylphosphoryl-enzyme complex, which, under most conditions, is stable. Examples of serine proteases which react with DIPF include, but are not limited to, trypsin, elastase, thrombin, and subtilisin. Kallikrein activity in an immunoglobulin preparation or plasmin activity in a prourokinase preparation are examples of common serine protease contamination.
Diisopropylfluorophosphate has been shown to be an effective inhibitor for many serine proteases. However, there are problems associated with the use of diisopropyl fluorophosphate. First, diisopropyl fluorophosphate is a hazardous chemical as identified by the National Research Council Committee on Hazardous Substances in the laboratory. Second, the inhibitory effect of diisopropyl fluorophosphate diminishes with time due to hydrolysis, causing eventual release of the active enzyme.
Affinity chromatography is a biochemical technique used to isolate certain proteins from complex mixtures such as blood or urine. This technique is based on the biochemical attraction of these proteins for certain molecules, commonly referred to ligands. Specific proteins are highly attracted to ligands such as the polysaccharide agarose, and strong bonds are formed when these proteins are allowed to contact the ligand molecules. The other proteins and substances within the mixture are not attracted to the ligand and may be separated from the protein/ligand complex by filtration, centrifugation or chromatographic techniques.
One example of an affinity-type separation is an affinity chromatography column. Ligand molecules that are specific for a particular type of protein, are bound to a support that is packed into a column. The mixture to be resolved, including the protein to be isolated, is introduced into one end of the column. The protein to be isolated becomes bound to the ligand as it passes through the column while the remaining substances in the mixture pass through the column without interaction with the ligand. The substances that were not bound to the ligand exit through the opposite end of the column by the force of gravity or by the use of a high-pressure pump. The ligand molecules remain stationary within the chromatography column during the separation procedure. The bound protein is then eluted from the column by rinsing the column with a solution which is capable of releasing the protein from the ligand.
Affinity gels can also be formed into a slab or rod for the electrophoretic separation of proteins based on relative ionic charge. Typical of these types of gels are polyacrylamide gels.
Thus, isolation of a particular protein from a biological sample by affinity chromatography depends upon the high affinity of the selected protein for the ligand and the lack of affinity between the other constituents of the sample and the ligand.
Consequently, there is a need for a safe and effective method of removing serine proteases from a biological sample to improve protein purification and to increase the storage potential for isolated protein samples.